The eukaryotic nucleus is a highly ordered structure that carries out an assortment of complex functions. In order for the nuclear tasks to be carried out properly, they must be temporally and spatially coordinated amongst themselves and with respect to the other functions of the cell. We are investigating how cells send and interpret signals that coordinate these activities with each other and with the cell cycle. Among the proteins that are thought to be important for maintaining nuclear integrity, two proteins that appear to be essential for the spatial and temporal order of the nucleus are Ran and RCC1. These two proteins interact enzymatically with each other, and they are required for almost every nuclear process including RNA transcription and processing, nuclear transport, DNA replication, and cell cycle control. Ran is a small, very abundant Ras-like GTPase that is mainly nuclear, while RCC1 is a chromatin-bound protein that acts as a guanine nucleotide exchange factor (GEF) for Ran. Our goal is to understand RCC1 and Ran at a molecular level and to discover how they interact with other cellular components that regulate cell cycle progression and interphase nuclear activities. We have studied RCC1 and Ran in biochemical assays using purified components and in Xenopus laevis egg extracts. We have also begun to purify other proteins that interact with RCC1 and Ran from Xenopus egg extracts. We have thereby discovered a number of novel proteins that may be important for the activity of this GTPase pathway. This work is complemented by an analysis of chemical agents that bypass S phase cell cycle control in mammalian cells and by a search for the molecular target(s) of these agents. In particular, we are examining the regulation of mRNA stability by checkpoint regulatory systems. We have found a number of candidate mRNAs whose stability appears to be controlled by the replication state of the nuclear DNA and we are currently in the process of characterizing these messages further.